PROJECT SUMMARY Innate lymphoid cells (ILCs) are a recently described class of lymphocytes that are critical for defense against a variety of pathogens, and their dysfunction has been associated with several inflammatory pathologies. Thus, understanding how ILCs are regulated is key to developing treatments for a wide array of infectious and inflammatory diseases. ILCs have been divided into three groups (ILC1, ILC2 and ILC3) based on their cytokine secretion profiles and functions. Recently, multiple factors have been identified to play critical roles in the development or function of ILCs; yet, most of these factors are functionally relevant in more than one group of ILCs. These results suggest that additional regulatory mechanisms unique to each ILC group might exist and that they could be critical for the functional differentiation of the different ILC subsets. Addressing this gap of knowledge will not only lead to a better understanding of the pathways that control the development, homeostasis and functions of each ILC group, but it might also uncover new antimicrobial or immunomodulatory therapeutic targets. Long noncoding RNAs (lncRNAs) are a novel class of noncoding RNAs that are now considered key regulators of gene expression programs. Importantly, lncRNAs are expressed in a more cell type-specific manner than protein coding genes, suggesting that they might play central roles in establishing gene expression programs that are unique to specific cell populations in vivo. However, whether lncRNAs can regulate the functional specification of individual ILC groups has not yet been examined. Thus, we hypothesize that there are lncRNAs that are specifically expressed in each group of ILCs and that they play critical roles in establishing the transcriptional programs unique to each ILC subset. To address this hypothesis, we combined a novel bioinformatic workflow to identify lncRNAs expressed in each ILC subset with the CRISPR/Cas9 system to rapidly generate knockout mouse lines. In our preliminary studies, we identified a lncRNA (termed Rroid) that is specifically expressed in mouse and human ILC1s, but not in ILC2s or ILC3s. Importantly, ablation of Rroid in the mouse leads to profound defects in the numbers and function of ILC1s. Furthermore, we found that Rroid controls the expression of the transcriptional repressor Id2 in ILC1s and that dysregulation of Id2 is the main cause for ILC1 dysfunction in Rroid-deficient mice. In here, we propose (i) to determine what aspects of ILC1 development, maturation or function Rroid regulates (Aim 1) and (ii) to establish the molecular mechanism by which Rroid regulates Id2 expression in ILC1s (Aim 2). Altogether, the completion of the proposed studies will fundamentally advance our knowledge of lncRNA regulation of the immune system and define the role of a novel lncRNA in ILC1s. Furthermore, since ILC1s are essential for cancer immunosurveillance and defense against intracellular pathogens, Rroid might be a novel therapeutic target for the treatment of these prevalent diseases. Finally, we will establish all the technical elements for future efforts that will aim to identify highly relevant lncRNAs in each of the ILC subsets.